Gregory G. Lennon

Crx, a Novel Otx-like Paired-Homeodomain Protein, Binds to and Transactivates Photoreceptor Cell-Specific Genes

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Sequence and Expression of a Candidate for the Human Secretor Blood Group α(1,2)Fucosyltransferase Gene (FUT2) HOMOZYGOSITY FOR AN ENZYME-INACTIVATING NONSENSE MUTATION COMMONLY CORRELATES WITH THE NON-SECRETOR PHENOTYPE

Synthesis of soluble A, B, H, and Lewis b blood group antigens in humans is determined by the Secretor (Se) (FUT2) blood group locus. Genetic, biochemical, and molecular analyses indicate that this locus corresponds to an α(1,2)fucosyltransferase gene distinct from the genetically-linked H blood group α(1,2)fucosyltransferase locus. The accompanying paper (Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D.(1995) J. Biol. Chem. 270, 4632-4639) describes the molecular cloning and mapping of two human DNA segments that are physically linked to, and cross-hybridize with, the H locus. We present here an analysis of these two new DNA segments. One of these, termed Sec1, is a pseudogene, because translational frameshifts and termination codons interrupt potential open reading frames that would otherwise share primary sequence similarity with the H α(1,2)fucosyltransferase. The other DNA segment, termed Sec2, predicts a 332-amino acid-long polypeptide, and a longer isoform, that share 68% sequence identity with the COOH-terminal 292 residues of the human H blood group α(1,2)fucosyltransferase. Sec2 encodes an α(1,2)fucosyltransferase with catalytic properties that mirror those ascribed to the Secretor locus-encoded α(1,2)fucosyltransferase. Approximately 20% of randomly-selected individuals were found to be apparently homozygous for an enzyme-inactivating nonsense allele (Trp143 → ter) at this locus, in correspondence to the frequency of the non-secretor phenotype in most human populations. Furthermore, each of six unrelated non-secretor individuals are also apparently homozygous for this null allele. These results indicate that Sec2 corresponds to the human Secretor blood group locus (FUT2) and indicate that homozygosity for a common nonsense allele is responsible for the non-secretor phenotype in many non-secretor individuals.

Molecular Cloning of a Human Genomic Region Containing the H Blood Group α(1,2)Fucosyltransferase Gene and Two H Locus-related DNA Restriction Fragments ISOLATION OF A CANDIDATE FOR THE HUMAN SECRETOR BLOOD GROUP LOCUS

We have used the human H blood group α(1,2)fucosyltransferase (FUT1) cDNA to screen chromosome 19 cosmid libraries in a search for the human Secretor (Se) blood group gene (FUT2). One cosmid has been isolated that contains two distinct segments that cross-hybridize with FUT1. We have assembled a 100-kilobase (kb) cosmid contig, localized to 19q13.3, encompassing FUT1 and the two FUT1-related sequences, termed Sec1 and Sec2, for Secretor candidate 1 and 2. Sec1 and Sec2 are separated by 12 kb and are 65.5 kb and 35 kb apart, respectively, from the FUT1 gene. We used a cosmid-dependent direct cDNA selection method to clone a cDNA corresponding to a transcript that emanates from Sec2. This cDNA detects a 3.35-kb transcript in human tissues known to express the Se locus. Together with sequence and expression data reported in the accompanying article (Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B.(1995) J. Biol. Chem. 270, 4640-4649), these data demonstrate that Sec2 corresponds to the human Se blood group locus (FUT2). Our results furthermore define the physical relationship between the H and Se loci and confirm a hypothesis that these two loci represent distinct but closely linked α(1,2)fucosyltransferase genes.

Identification of seven new human MHC class I region genes around the HLA-F locus.

Using cDNA hybridization selection techniques, we identified seven new genes in a 280 kilobase YAC coveting theHLA-F locus. The new genes were mapped back to the YAC by a combination of optical restriction mapping and pulse field gel electrophoresis. Northern analysis of individual clones demonstrated the presence of either different mRNA sizes or different expression patterns. Two of the cDNA clones were expressed only in lymphoid cell lines: one in Jurkat cells (T cell) and another in JY cells (B cell). All the genes lacked sequence similarity to any known classical and non-classical major histocompatibility complex (MHC) class I genes, indicating that theMHC class I region has more functions than anticipated. Of the seven new genes, one is highly similar (97%) to mouse 60S ribosomal protein, and another is homologous to diubiquitin proteins. Of the two G-coupled receptor-like cDNAs, one was fully sequenced and found to be an olfactory receptor-like gene. The study strengthens evidence that theMHC complex not only plays a key role in the immune system, but also contributes to non-immunological functions.

From genes to proteins: high-throughput expression and purification of the human proteome.

The development of high‐throughput methods for gene discovery has paved the way for the design of new strategies for genome‐scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high‐throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high‐throughput expression system for protein production.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR

AbstractBackground: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. Methods and Results: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. Conclusion: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Gene database for the fission yeast Schizosaccharomyces pombe

SummaryAs an aid to the fission yeast genome project, we describe a database for Schizosaccharomyces pombe consisting of both genetic and physical information. As presented, it is therefore both an updated gene list of all the nuclear genes of the fission yeast, and provides an estimate of the physical distance between two mapped genes. Additionally, a field indicates whether the sequence of the gene is available. Currently, sequence information is available for 135 of the 501 known genes.

Characterization of the human neurocan gene, CSPG3.

Neurocan is a chondroitin sulfate proteoglycan thought to be involved in the modulation of cell adhesion and migration. Its sequence has been determined previously in rat and mouse (Rauch et al., 1992. Cloning and primary structure of neurocan, a developmentally regulated, aggregating, chondroitin sulfate proteoglycan of the brain. J. Biol. Chem. 267, 19536-19547; Rauch et al., 1995. Structure and chromosomal location of the mouse neurocan gene. Genomics 28, 405-410). We describe here the complete coding sequence of the human neurocan mRNA, known as CSPG3, as well as mapping data, expression analysis, and genomic structure. A cDNA known as CP-1 was initially sequenced as part of a gene discovery project focused on characterizing chromosome 19-specific cDNAs. Sequence homology searches indicated close homology to the mouse and rat proteoglycan, neurocan (GenBank accession Nos X84727 and M97161). Northern analysis identified a brain-specific transcript of approx. 7.5kb. A longer cDNA clone, GT-5, was obtained, fine-mapped to the physical map of chromosome 19 by hybridization to a chromosome-specific cosmid library, and sequenced. Full coding sequence of the mRNA indicates a 3963bp open reading frame corresponding to a 1321 amino acid protein, similar to the protein length found in mouse and rat. The amino acid sequence of human neurocan shows 63% identity with both the mouse and rat sequences. Finally, genomic sequencing of a cosmid containing the complete neurocan gene was performed to determine the genomic structure of the gene, which spans approx. 41kb, and is transcribed in the telomere to centromere orientation.

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19.

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.

The fucosyltransferase locus FUT1 maps distal to apolipoprotein loci E and C2 on human chromosome 19

The location of the fucosyltransferase locus FUT1 relative to the apolipoprotein E/C2 loci on human chromosome 19 has remained unclear. We determined by a combination of physical mapping and fluorescence in situ hybridization that this fucosyltransferase gene maps distal to the apolipoprotein loci.